A large number of transition metal complexes, particularly those of the platinum class of metals with d.sup.6 electronic configuration with a class of ligands generally referred to as .alpha.-diimines, have unique optical, electrochemical, photochemical and photophysical properties. These properties, which are based on efficient and facile electron-transfer and energy-transfer processes, are responsible for numerous applications in the areas of photocatalysis, electrocatalysis and photoelectrolysis [Kalyanasundram, Coordination Chemistry Reviews, 46, 159 (1982); Kalyanasundaram, Gratzel and Pelizzetti, Coordination Chemistry Reviews, 69, 57 (1986); Serpone, Photochemical Energy Conversions, Elsevier, Amsterdam, 1989; and Collins and Sauvage, Coordination Chemistry Reviews, 93, 245 (1989)]. A large number of ruthenium(II) and osmium(II) polypyridine complexes have been synthesized and evaluated for a variety of applications utilizing their photoactive and electroactive properties [Seddon and Seddon, The Chemistry of Ruthenium, Elsevier, Amsterdam, 1984; Juris, Balzani, Barigelletti, Campagna, Besler, and Von Zelevsky, Coordination Chemistry Reviews, 84, 85 (1988); and Balzani, Juris and Scandola, Homogeneous and Heterogeneous Catalysis, Reidel Publishing Corporation, New York, 1990]. Numerous such complexes have been used to prepare chemically-modified electrodes, which can be applied as electrocatalytic and electrochemical sensors (Murray in Bard (ed.), Electrochemical Chemistry, Vol. 13, M. Dekker, New York, 1984, p. 1 and Skotheim (ed.), Handbook of Conducting Polymers, M. Dekker, New York, 1986). Many of these complexes emit light strongly at room temperature and their luminescence can be quenched by a variety of reagents including molecular oxygen. The luminescence properties of this class of metal complexes enable their use as molecular sensing elements in fiber-optic chemical sensors and biosensors [Wolfbeis, Fiber-Optic Chemical Sensors and Biosensors, CRC Press, Boston, 1991]. Fiber-optic chemical sensors (the general term `sensor` will be used both for chemical sensors and biosensors in this document) offer advantages to electrochemical sensors for monitoring and determining chemical and biochemical analytes. Of various optical methods employed for chemical analysis, those based on luminescence (emission) spectroscopy are considered particularly attractive because of their high sensitivity and specificity. Both inorganic and organic luminescent materials can be used as `sensing elements`, `molecular probes`, `reporters` or `indicators` for remote quantification of various physical parameters (e.g. temperature, pH, pO.sub.2, and pCO.sub.2) and chemical (e.g. ethanol and methanol) and biochemical analytes (e.g. glucose, creatinine and cholesterol).
A variety of metal-organic compounds of a number of transition metals and lanthanides are known to be intensely luminescent. Luminescent transition metal complexes, especially of d.sup.6 platinum metals such as ruthenium, osmium, rhenium, rhodium and iridium with .alpha.-diimine type ligands (for example, 2,2'-bipyridine, 1,10-phenanthroline, terpyridine and their substituted derivatives) exhibit very desirable features in terms of their optical spectra, excited state lifetimes and luminescence quantum yields, making them very attractive luminophores for fiber-optic sensors. Ruthenium(II) and rhenium(I) carbonyl halide-polypyridine complexes exhibit intense visible absorption, which is important for sensitivity, simplicity of sensor design and adaptability to a variety of excitation sources. Their excited states have long lifetimes and high luminescence quantum yields that are independent of the excitation wavelength. The absorption and emission characteristics of metal-polypyridine complexes can be fine-tuned by varying substituents on the polypyridine ligand or by changing the metal atoms. Fine-tuning of optical, photophysical and redox properties of the metal complexes can provide tailor-made luminophores for fabricating a variety of fiber-optic sensors for environmental, oceanographic, industrial, biotechnological and biomedical applications. For instance, luminescence-based oxygen sensors for oceanographic applications should contain a luminophore whose emission maximum does not lie in the 660-700 nm range, where emission of chlorophyll can interfere with the emission of the molecular probe of the sensor. Oxygen sensors designed for invasive biomedical applications have to take into account the properties of various luminescent materials present in blood, which can interfere with emission signal or quench the luminescent state of the luminophore.
A large number of ruthenium(II), osmium(II) and rhenium(I) carbonyl halide-polypyridine complexes [Sacksteder, Zipp, Brown, Streich and Demas, Inorganic Chemistry, 29, 4335 (1990)] exhibit a good combination of desirable properties, making them suitable luminophores in various sensor applications [Demas and DeGraff, Proceedings SPIE, 1172, 216 (1989)]. None of these metal-polypyridine complexes are ideal for commercial applications for various reasons, including their tendency for ligand substitution (photolability) and difficulty for chemical immobilization in polymeric matrices. For long-term stability it is essential that the luminophore be chemically immobilized on an insoluble polymeric support through the formation of a covalent bond [Seitz, CRC Critical Reviews in Analytical Chemistry, 19, 135, 1988].
Several methods can be employed for immobilization of chemical and biochemical species. The most effective immobilization procedure is one in which a chemical bond is formed between a polymer and the species to be immobilized. Immobilization very often results in attenuation of various characteristics of a reactive species. However, attachment of low molecular weight luminophores to polymeric supports can result in less severe attenuation of vital luminophore characteristics [Carraway, Demas, DeGraff and Bacon, Analytical Chemistry, 63, 337 (1991)].
A number of sensing elements [Marsoner, Kroneis and Wolfbeis, European Patent Application, EP 109,959 (1984); Barnikol and Burkhard, German Offen. DE 3,320,752 (1983); Hsu and Heitzmann, U.S. Pat. No. 4,712,865 (1987); Kung, Vogelhut, U.S. Pat. No. 4,560,534 (1985)] and methods, schemes and apparatus [Buckles, PCT International Application 8,100,912 (1981); Bacon and Demas, French Demande FR 2,538,550 (1984); Bacon and Demas, U.S. Pat. No. 5,030,420 (1991); Klainer, Walt and Gottlieb, International Application Number PCT/US88/00041 (1988); Wagner, U.S. Pat. No. 5,001,054 (1991); Higgins, Hill, Plotkin, U.S. Pat. No. 4,711,245 (1987)] have been devised and evaluated. Polymer-catalyst based transducers used by King and Vogelhut for the determination of glucose in whole blood do not make use of platinum metal-polypyridine complexes and are only designed for conductometric measurements. Bacon and Demas [French Demande FR 2,538,550 (1984) and U.S. Pat. No. 5,030,420 (1991)] described the use of sensing elements based on platinum metal-polypyridine complexes which have no possibility of chemical or covalent attachment to polymeric matrices. These metal complexes could only be used as physical mixtures with polymers, producing inhomogeneous distribution of the sensing elements and rapid leaching of the luminophore into the analyte solution. Wagner [U.S. Pat. No. 5,001,054 (1991)] proposed a method based on the use of a conjugate of glucose and a fluorescent dye coated onto an optical fiber for monitoring the glucose level in body fluid. This sensor is not expected to be durable because the dye is not covalently immobilized. Klainer, Walt and Gottlieb [International Application Number PCT/US88/00041 (1988)] have also described fiber optic sensing devices for measuring chemical or physiochemical parameters of a body fluid or tissue. These devices are based on polymeric photoactive dyes employed as sensing elements for pH and oxygen sensors.
Despite considerable efforts, no metal-organic luminophore has yet demonstrated the possibility of chemical immobilization while maintaining most of their useful optical, photophysical and photochemical characteristics. Chemically immobilized luminophores can be cast in ultrathin films containing evenly distributed sensing material. In fiber-optic sensors for continuous monitoring of chemical and biochemical analytes, the time to establish equilibrium between analyte and sensing element is of crucial importance. Ultrathin films containing immobilized luminophores can be used to produce fiber-optic sensors with very short response times.
Immobilization processes involve two components: (a) polymeric supports and (b) immobilization methods. The choice of polymeric supports is generally governed by its permeability for the analyte, stability, availability, suitability for sensing element immobilization and its compatibility with the sample and various materials used in the fabrication of optrodes [Hodge and Sherrington, Polymer-supported Reactions, J. Wiley, New York, 1980 and R. B. Seymour and Marks, Applications of Polymers, John Wiley & Sons, New York, 1988]. The choice of the right material is very important because the nature of the polymer has a pronounced effect on sensor performance. For example, the response time is governed by the diffusion coefficients of gases or liquids and the quenching efficiency by the solubility of the gas in the polymer. Among the variety of polymers evaluated for immobilization, siloxanes are unique in having permeability for most gases as well as excellent biocompatibility and optical and mechanical properties [Robb, Thin Silicone Membranes-Their Permeation Properties and Some Applications, Annals New York Academy of Science, 146, 119 (1986) and Anderson, Arkles and Larson, Silicone Compounds Register and Reviews, Petrarch Systems, Bristol, Pa., 1987]. Numerous silicone polymers are commercially available and allow for easy manufacture of membranes, emulsions, suspensions and coatings. Other hydrophobic polymeric supports include poly(vinyl chloride), poly(ethylene), poly(tetrafluoroethylene) and poly(styrene). Except for poly(styrene), most of other polymers are modified with difficulty. Even polystyrene is not very suitable for immobilization of luminophores, as it can interfere with their luminescence properties. A variety of hydrophilic supports characterized by a large number of hydrogen bonding groups, such as OH or NH.sub.2, or by the presence of a number of charged groups such as COO.sup.-- or SO.sub.3.sup.-- in the polymer chain, are also known. Typical examples include polysaccarides (celluloses), polyacrylates, poly-acrylamides, polyglycols, and a variety of so-called hydrogels. Generally, these polymers are easily penetrated by aqueous solutions and they have limited compatibility with hydrophobic polymers. Several polymeric supports with mixed hydrophilic and hydrophobic groups are also known. Typical examples include ion-exchange materials such as poly(acrylonitrile-methylsulfonate) copolymers, polysulfones, and partially fluorinated polyethylene (Nafion).
Several immobilization techniques are available for combining `sensing reagents` with polymeric supports [Hodge and Sherrington, Polymer-supported Reactions in Organic Synthesis, John Wiley, New York, 1980 and Sharma, Bailey and Messing, Immobilized Bio-materials: Techniques and Applications, Angew Chemie, International Edition, English Translation, 21, 837 (1982)]. Three immobilization methods, namely, chemical covalent, physical and electrostatic techniques, are commonly used for the preparation of immobilized sensing molecules. Mechanical or physical immobilization or encapsulation involves adsorption and inclusion of molecules in polymer matrices. This is the simplest and therefore the least expensive way of immobilization. However, in this type of immobilization there is no bonding between the sensing reagent and the polymeric support and the immobilized luminophores can leach out. Electrostatic immobilization uses rigid polymeric supports with charged groups such as sulfonic (sulfonated polystyrene) or quaternized ammonium groups capable of binding electrostatically to molecules of opposite charge. For example, sulfonated polystyrene can bind a variety of cations with varying strength. Numerous ion-exchange materials of varying binding strengths are commercially available [Bishop, Indicators, Pergamon Press, New York, 1972; Zhujun and Seitz, Analytica Chimica Acta, 160, 47, 1984]. The ease of execution and the degree of reproducibility of electrostatic immobilization is also decreased by non-homogeneous distribution of sensing materials and their bleeding on long-term use.
Current methods for immobilizing luminescent metal complexes in polymer matrices are not adequate for practical applications, such as fiber-optic sensors for oxygen determination. The method of Bacon and Demas [French Demande FR 2,538,550 (1984) and U.S. Pat. No. 5,030,420 (1991)] produces materials in which the luminophore is physically mixed with the polymer matrix. The resulting polymer films lack durability because the luminophore can be leached from the polymer matrix. S. L. Buell, et al. [J. Phys. Chem. 1983, 87, 4675-4681] have described electrostatically bonded luminophores combining ruthenium polypyridine complexes with ion exchange resins. While somewhat more durable, these materials are subject to leaching of the luminophore by displacement with ions in the analyte solution. Also, the polymer matrix cannot be varied easily because ion-exchange properties are required for this method of immobilization. Finally, K. T. Potts, et at. [Macromolec. 1988, 21, 1985-1991] have described polystyrene copolymers containing covalently bound terpyridyl ligands and Ru(tpy).sub.2 X.sub.2 complexes. While these platinum-metal complexes are covalently bound to the polymer matrix, they are not described as luminescent. Low luminescence quantum yields are expected for such materials because of interactions between the excited luminophore and the polystyrene matrix, as mentioned previously. Moreover, polystyrene has inferior physical properties (e.g. permeability and polarity) for sensor applications in aqueous solutions and the polymer matrix cannot be varied easily with this method of luminophore immobilization.